Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.

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Progesterone receptor in the chick oviduct: an immunohistochemical study with antibodies to distinct receptor components.

The Journal of Cell Biology ◽

10.1083/jcb.99.4.1193 ◽

1984 ◽

Vol 99 (4) ◽

pp. 1193-1201 ◽

Cited By ~ 114

Author(s):

J M Gasc ◽

J M Renoir ◽

C Radanyi ◽

I Joab ◽

P Tuohimaa ◽

...

Keyword(s):

Progesterone Receptor ◽

Polyclonal Antibodies ◽

Smooth Muscles ◽

Immunoperoxidase Technique ◽

Cell Nuclei ◽

Receptor Complexes ◽

B Subunit ◽

Chicken Oviduct ◽

A Subunit ◽

Immunohistochemical Studies

We performed immunohistochemical studies of chicken oviduct after different fixation procedures, by using antibodies against the progesterone receptor: polyclonal antibodies IgG-G3 against the "8S" form (an oligomere containing progesterone-binding and nonprogesterone-binding units), polyclonal antibodies IgG-RB against the progesterone-binding B subunit, and monoclonal BF4 against the non-progesterone-binding 90,000-mol-wt protein component. Chickens were immature animals with or without estrogen priming, and with or without progesterone treatment. The antibodies were revealed by means of an immunoperoxidase technique that used the avidin-biotin-peroxidase complex, and controls were performed by presaturation of antibodies with the purified 8S-progesterone receptor, the B subunit, and 90,000-mol-wt protein. The progesterone receptor was detected not only in well-characterized target tissues, i.e., in glands and luminal epithelium, but also in stromal cells (some displayed the strongest reaction), in mesothelium, and in fibers of smooth muscles. Only in cell nuclei, whether or not the animals received an injection of progesterone was an antigen revealed corresponding to the B subunit (and/or to the A subunit, because there is immunoreactivity of IgG-RB with both hormone-binding subunits A and B). The 90,000-mol-wt protein was revealed in both cytoplasm and nuclei. These immunohistological data suggest that the concept of steroid action that necessarily involves the original formation of the hormone-receptor complexes in the cytoplasm before translocation to the nucleus, may have to be revised.

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Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.869-876.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 869-876

Author(s):

D F Smith ◽

W P Sullivan ◽

T N Marion ◽

K Zaitsu ◽

B Madden ◽

...

Keyword(s):

Progesterone Receptor ◽

Simian Virus 40 ◽

Immunoblot Analysis ◽

Simian Virus ◽

Rabbit Reticulocyte Lysate ◽

Striking Similarity ◽

Peptide Fragments ◽

Receptor Complexes ◽

Reticulocyte Lysate ◽

A Cell

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.

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Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6804 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6804-6812 ◽

Cited By ~ 212

Author(s):

D F Smith ◽

L Whitesell ◽

S C Nair ◽

S Chen ◽

V Prapapanich ◽

...

Keyword(s):

Progesterone Receptor ◽

Binding Capacity ◽

Biological Significance ◽

Binding Agent ◽

Complex Dynamic ◽

Intact Cells ◽

Receptor Complexes ◽

Assembly Pathway ◽

And Function

The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.

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Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.869 ◽

1993 ◽

Vol 13 (2) ◽

pp. 869-876 ◽

Cited By ~ 192

Author(s):

D F Smith ◽

W P Sullivan ◽

T N Marion ◽

K Zaitsu ◽

B Madden ◽

...

Keyword(s):

Progesterone Receptor ◽

Simian Virus 40 ◽

Immunoblot Analysis ◽

Simian Virus ◽

Rabbit Reticulocyte Lysate ◽

Striking Similarity ◽

Peptide Fragments ◽

Receptor Complexes ◽

Reticulocyte Lysate ◽

A Cell

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.

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Mutation of Hip’s Carboxy-Terminal Region Inhibits a Transitional Stage of Progesterone Receptor Assembly

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.944 ◽

1998 ◽

Vol 18 (2) ◽

pp. 944-952 ◽

Cited By ~ 42

Author(s):

Viravan Prapapanich ◽

Shiying Chen ◽

David F. Smith

Keyword(s):

Progesterone Receptor ◽

Receptor Complexes ◽

Multiple Components ◽

C Terminus ◽

Dependent Inhibition ◽

Hip Function ◽

Carboxy Terminal Region ◽

Dose Dependent Inhibition ◽

Carboxy Terminal ◽

Receptor Assembly

ABSTRACT Steroid receptor complexes are assembled through an ordered, multistep pathway involving multiple components of the cytoplasmic chaperone machinery. Two of these components are Hsp70-binding proteins, Hip and Hop, that have some limited homology in their C-terminal regions, outside the sequences mapped for Hsp70 binding. Within this region of Hip is a DPEV sequence that occurs twice; in Hop, one DPEV sequence plus a partial second sequence occurs. In an effort to better understand Hip function as it relates to assembly of progesterone receptor complexes, the DPEV region of Hip was targeted for mutations. Each DPEV sequence was mutated to an APAV sequence, singly or in combination. The combined mutation, APAV2, was further combined with a deletion of Hip’s tetratricopeptide repeat region that is required for Hsp70 binding or with a deletion of Hip’s GGMP repeat. An additional mutant was prepared by truncation of Hip’s DPEV-containing C terminus. By comparing interactions of various Hip forms with Hsp70, it was determined that mutation of the DPEV sequences created a dominant inhibitory form of Hip. The mutant Hip-Hsp70 complex was not prevented from interacting with progesterone receptor, but the mutant caused a dose-dependent inhibition of receptor assembly with Hsp90. The behavior of the Hip mutant is consistent with a model in which Hip and Hop are required to facilitate the transition from an early receptor complex with Hsp70 into later complexes containing Hsp90.

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Inhibition of TMEM16A impedes embryo implantation and decidualization in mice

Reproduction ◽

10.1530/rep-18-0197 ◽

2018 ◽

Cited By ~ 2

Author(s):

Qianrong Qi ◽

Yifan Yang ◽

Kailin Wu ◽

Qingzhen Xie

Keyword(s):

Progesterone Receptor ◽

Stromal Cells ◽

Embryo Implantation ◽

Mouse Uterus ◽

Endometrial Stromal Cells ◽

Uterine Endometrium ◽

Molecule Inhibitor ◽

Pathway Inhibition ◽

And Function ◽

Reproductive Processes

Recent studies revealed that TMEM16A is involved in several reproductive processes, including ovarian estrogen secretion and ovulation, sperm motility and acrosome reaction, fertilization, and myometrium contraction. However, little is known about the expression and function of TMEM16A in embryo implantation and decidualization. In this study, we focused on the expression and regulation of TMEM16A in mouse uterus during early pregnancy. We found that TMEM16A is up-regulated in uterine endometrium in response to embryo implantation and decidualization. Progesterone treatment could induce TMEM16A expression in endometrial stromal cells through progesterone receptor/c-Myc pathway, which is blocked by progesterone receptor antagonist or the inhibitor of c-Myc signaling pathway. Inhibition of TMEM16A by small molecule inhibitor (T16Ainh-A01) resulted in impaired embryo implantation and decidualization in mice. Treatment with either specific siRNA of Tmem16a or T16Ainh-A01 inhibited the decidualization and proliferation of mouse endometrial stromal cells. In conclusion, our results revealed that TMEM16A is involved in embryo implantation and decidualization in mice, compromised function of TMEM16A may lead to impaired embryo implantation and decidualization.

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